Cosmetic and dermopharmaceutical compositions for skin prone to acne

ABSTRACT

The invention relates to the use of an extract of olive leaves ( Olea europaea ) which is titrated in oleanolic acid and which may or may not be associated with a  Larrea divaricata  extract which is titrated in nordihydroguaiaretic acid (NDGA). Said products are intended for all types of cosmetic and dermopharmaceutical compositions for all forms of skin care, for moisturizing and anti-inflammatory purposes and, in particular, for the prevention and treatment of skin prone to acne.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/817,670, filed on Apr. 2, 2004, which is a continuation-in-part ofPCT Patent Application No. PCT/FR 02/03344, designating the U.S., filedOct. 1, 2002, which was published in French, which claims priority fromFrench Patent Application No. FR 01/12802, filed Oct. 3, 2001, thedisclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Various forms of acne exist. These include: juvenile, which may emergeas of age 9 years, with no premonitory sign of puberty, and whichaffects approximately 70% of adolescents of both genders; adult, which,outside of the consequences of hormonal contraceptive treatment,corresponds, in fact, in most cases, to juvenile acne persisting in theabsence of treatment of after inappropriate treatment; drug-related, themain causes of which are vitamin B12, systemic or local corticosteroids,iodine- or bromine-based preparations, androgens and androgenicprogestogen contraceptive pills; neonatal, due to maternal androgens,and for which recovery is spontaneous; excoriated, of young girls, whichis an exacerbation of normal and classic juvenile acne due to almostobsessive touching of acne spots.

Thus, most forms of acne derive more or less directly from juvenileacne. Acne is, in all likelihood, a multifactorial disease which can besummarized by the following triad: hyperseborrhea+disorder ofkeratinization of the pilosebaceous canal+a microbial factor.

Hyperseborrhea

There is no acne without hyperseborrhea and, broadly speaking, acne isproportional to the degree of seborrhea. Sebum secretion is underhormonal control and constitutes one of the best indicators of androgenlevels explaining the emergence of acne at the time of puberty, duringwhich a physiological hormonal explosion occurs.

The androgen hormone most involved in acne is testosterone. Gonadaltestosterone circulates in the body in a protein-bound form and onlyfree testosterone enters the target cell: the sebaceous gland. There, anenzyme, 5α-reductase, converts testosterone to its metabolite,dihydrotestosterone (DHT), which stimulates the synthesis of nuclearproteins. The more DHT which reaches the nucleus, the greater theincrease in sebaceous gland size and proliferation. The glands fill withlipids and, since the sebaceous gland is a holocrine secretory gland,the greater the quantity of excreted sebum.

Keratinization Disorder of the Pilosebaceous Canal

This point constitutes a sine qua non condition for the occurrence ofacne. The epithelium bordering the pilosebaceous canal atinfra-infundibulum level then forms a large quantity of abnormallykeratinized cells, a large quantity of keratin and a particularlysparingly soluble intercellular ground substance. At infra-infundibulumlevel, this creates a compact plug which prevents expulsion of thesebum: this is the microcyst or closed comedo stage, the true elementarylesion of acne. The microcyst can then evolve in 2 ways: either thekeratin and sebum continue to provoke swelling of the microcyst forcingdilation of the acro-infundibulum, thus forming an open comedo or commonblackhead, with no real seriousness; or the wall of the microcystruptures with irruption into the dermis of sebum, keratin and free fattyacids. An inflammatory reaction occurs, which is exacerbated by the lastcomponent in the etiology of acne.

Microbial Factor

Normally, in the pilosebaceous follicle, 3 types of microorganism exist:a yeast: Pityrosporum ovale, whose pathogenic role in acne appears nil,a non-pathogenic white Staphylococcus, an anaerobic bacteria,diphtheroid and Gram-positive: Corynebacterium acnes (formerly calledPropionibacterium acnes), which synthesizes a lipase able to hydrolyzethe triglycerides of sebum into free fatty acids that are irritants forthe dermis. This microorganism is also involved in the local productionof protease, hyaluronidase and neuraminidase, which exacerbate theinflammatory process cited above.

The data presented above are derived from a review of the followingdocuments: F. Poli (1996): Acné prépubertaire, Le Concours Médical,118:905-908; P. Morel (1981), polycopié de dermato et vénéro, Le KremlinBicêtre: 161-179; François Daniel (1977): Dermatologie pratique, EditionDupuy-Compton Médical, Neuilly. Both during adolescence and adulthood,acne induces a negative self-image in people who have acne-prone skin.The cosmetic industry is thus within its scope when it proposes a newapproach to resolving the problems of acne-prone skins.

SUMMARY OF THE INVENTION

The present invention includes the use of oleanolic acid, pure orisolated or contained in titrated olive tree (Olea europaea) leafextract in an effective and/or sufficient quantity, in a cosmetic ordermopharmaceutical composition. These compositions may be intended toprevent and treat the symptoms of acne, hyperseborrhea and acne-proneskin. The oleanolic acid, whether as part of an extract or otherwise maybe used along with an effective and/or sufficient quantity ofnordihydroguaiaretic acid (pure or isolated or in the form of a titratedLarrea divaricata extract).

Cosmetic or dermopharmaceutical compositions intended to prevent andtreat the symptoms of acne, hyperseborrhea and to prevent symptoms inacne-prone skin containing an effective and sufficient quantity ofoleanolic acid or titrated Olea europaea leaf extract containing it.These cosmetic or dermopharmaceutical compositions may also contain aneffective and/or sufficient quantity of nordihydroguaiaretic acid or atitrated Larrea divaricata extract containing it. In a preferredembodiment of these cosmetic or dermopharmaceutical compositions, thequantity of oleanolic acid is between 1 and 10,000 ppm (0.0001 and 1%m/m), preferably between 10 and 1000 ppm (0.001 and 0.1% m/m). Thequantity of nordihydroguaiaretic acid is, if present, between 1 and10,000 ppm (0.0001 and 1% m/m), preferably between 10 and 1000 ppm(0.001 and 0.1% m/m).

A particularly preferred cosmetic and/or dermopharmaceutical compositionincludes a concentration of the oleanolic acid (used pure or in the formof Olea europaea extract) of between 5 and 50 ppm (0.0005 to 0.05% m/m)and a concentration of nordihydroguaiaretic acid (used pure or in theform of titrated Larrea divaricata extract) of between 1 and 100 ppm(0.0001 to 0.01% m/m).

Cosmetic or dermopharmaceutical compositions may include any otheringredient usually employed in the fields of cosmetics anddermopharmacy. These can include, without limitation, lipids includingextracted and/or synthetic lipids, gelling and viscosifying polymers,surfactants and emulsifying agents, any water- or fat-soluble activesubstance, plant extracts of all sorts, synthetic peptides, proteins,vitamins, tissue extracts, marine extracts, sunscreens and antioxidants.A particularly preferred composition includes OSMOCIDE® (marketed by theSederma company, France). These cosmetic or dermopharmaceuticalcompositions may be combined with other active substances and may beused in any pharmaceutical form employed in cosmetics or dermopharmacysuch as, without limitation, gels, O/W and W/O emulsions, milks,lotions, ointments, scalp lotions, shampoos, soaps, sticks and pencils,sprays and body oils.

The active substances may be used in the form of a solution, dispersionor emulsion, or encapsulated in carriers such as macro-, micro- ornanocapsules, liposomes or chylomicrons, or included in macro-, micro-or nanoparticles, or in microsponges, or adsorbed on powdered organicpolymers, talcs, bentonites and other inorganic carriers.

Oleanolic acid, pure or in the form of titrated olive tree (Oleaeuropaea) leaf extract, alone or in combination withnordihydroguaiaretic acid (pure or in the form of titrated Larreadivaricata extract), or combined with other active substances can beused for the preparation of a medicinal product used for any form ofmoisturizing or anti-inflammatory care of the skin and in the preventionand treatment of acne-prone skin.

The cosmetic or dermopharmaceutical compositions of the invention mayhave the active substances impregnated on any sort of textile, syntheticor natural fiber, wool or any material liable to be used for themanufacture of clothing or underclothing, active materials of any sort,wipes, patches, compresses, cottons, cotton buds, dressings,makeup-removal sponges, masks and any other carrier liable to come intodirect contact with the skin or scalp to enable continuous topicaldelivery.

DETAILED DESCRIPTION

It has been discovered that oleanolic acid and extracts of olive tree(Olea europaea) leaves titrated with respect to oleanolic acid content,are endowed with a strong inhibitory action on the enzyme, 5α-reductase,and thus constitute an important component in the treatment of thesymptoms of acne-prone skin. It has also been discovered that oleanolicacid and the extracts of olive tree leaves are endowed withantimicrobial activity against Corynebacterium acnes and Acinetobactercalcoaceticus. The use of oleanolic acid and olive leaf extracts toprevent and treat the symptoms of acne and acne-prone skin is thus new.

In addition, surprisingly, it has been discovered that a synergy isobtained by combining nordihydroguaiaretic acid or an extract of Larreadivaricata of known nordihydroguaiaretic acid content with oleanolicacid or extracts of olive tree leaves.

Nordihydroguaiaretic acid is an inhibitor of protein metabolism in theGolgi apparatus and thus plays a moderating role in cell growth. The useof nordihydroguaiaretic acid to slow the growth of the skin andappendages has been described in FR 2785804. It appears that combinationof oleanolic acid and nordihydroguaiaretic acid or extracts containingthose compounds strengthens the antimicrobial activity and decreaseshyperkeratinization concomitantly, thus enhancing prevention of thesymptoms of acne.

Anti-acne activity can be further reinforced if the cosmetic anddermopharmaceutical compositions containing said extracts are formulatedwith other appropriate compounds such as keratolytic, sebum-regulating,moisturizing and/or osmotic active substances (particularly those withspecific antimicrobial and anti-inflammatory activities).

The other constituents of the compositions may be any habitually ornewly used or usable ingredient in cosmetics and dermopharmacy.

Oleanolic acid combined or not combined with nordihydroguaiaretic acidand/or the plant extracts containing them may thus be advantageouslyused in the prevention and treatment of acne-prone skin since: oleanolicacid decreases hyperseborrhea by inhibiting 5α-reductase andnordihydroguaiaretic acid (NDGA) exerts an anti-inflammatory andantiproliferative effect on keratinocytes and reduceshyperkeratinization.

In cosmetic or dermopharmaceutical compositions, it may be advantageousto combine the extracts cited with other active substances in order tostrengthen their effect by an additive or synergistic effect of thevarious products or in order to combine the effect described in thispatent application with another beneficial physiological effect for theskin, mucous membranes, cutaneous appendages (hair and scalp), such asOSMOCIDE® (Sederma).

The oleanolic acid and, possibly, nordihydroguaiaretic acidconcentrations in the cosmetic or dermopharmaceutical product may varyfrom 1 to 10,000 ppm (0.0001 and 1%, m/m), preferably between 10 and1000 ppm (0.001 and 0.1% m/m). In combination with nordihydroguaiareticacid, preferably 5 to 50 ppm (0.0005 to 0.05% m/m) of oleanolic acid and1 to 100 ppm (0.0001 to 0.01% m/m) of nordihydroguaiaretic acid will beused. The concentrations of the more or less crude or purified extractsof olive tree leaves and L. divaricata used in the finished productsdepend on their respective oleanolic acid and nordihydroguaiaretic acidcontents. The final concentrations of the main active substances(oleanolic acid and nordihydroguaiaretic acid) are to lie within theranges cited.

Oleanolic acid pure or in the form of titrated olive tree (Oleaeuropaea) leaf extract, alone or in combination withnordihydroguaiaretic acid (pure or in the form of titrated Larreadivaricata extract) or combined with other active substances, may beused in any pharmaceutical form employed in cosmetics or dermopharmacy:O/W and W/O emulsions, milks, lotions, ointments, scalp lotions,shampoos, soaps, sticks and pencils, sprays and body oils, without thatlist being exhaustive.

Oleanolic acid pure or in the form of titrated olive tree (Oleaeuropaea) leaf extract, alone or in combination withnordihydroguaiaretic acid (pure or in the form of titrated Larreadivaricata extract), or combined with other active substances, may beused in the form of solutions, dispersions and emulsions, orencapsulated in carriers such as macro-, micro- or nanocapsules andliposomes or chylomicrons, or included in macro-, micro- ornanoparticles or in microsponges, or adsorbed on powdered organicpolymers, talcs, bentonites or other inorganic carriers.

Oleanolic acid pure or in the form of titrated olive tree (Oleaeuropaea) leaf extract, alone or in combination withnordihydroguaiaretic acid (pure or in the form of titrated Larreadivaricata extract) may be combined with any other usually employedingredient: extracted and/or synthetic lipids, gelling and viscosifyingpolymers, surfactants and emulsifying agents, all water- and fat-solubleactive substances, plant extracts of any sort, synthetic peptides,proteins, vitamins, tissue extracts, marine extracts, sunscreens andantioxidants.

Oleanolic acid pure or in the form of titrated olive tree (Oleaeuropaea) leaf extract, alone or in combination withnordihydroguaiaretic acid (pure or in the form of titrated Larreadivaricata extract) or combined with other active substances, may beused in cosmetic or dermopharmaceutical compositions for all skin care,moisturizing or anti-inflammatory, and in the prevention and treatmentof acne-prone skin.

Oleanolic acid pure or in the form of titrated olive tree (Oleaeuropaea) leaf extract, alone or in combination withnordihydroguaiaretic acid (pure or in the form of titrated Larreadivaricata extract) or combined with other active substances, may beincorporated in cosmetic compositions or dermopharmaceuticals for thepreparation of medicinal products used in moisturizing oranti-inflammatory care of the skin and in the prevention and treatmentof acne-prone skin.

Oleanolic acid, pure or in the form of titrated olive tree (Oleaeuropaea) leaf extract, alone or in combination withnordihydroguaiaretic acid (pure or in the form of titrated Larreadivaricata extract), or combined with other active substances, may beused to impregnate any sort of textile, synthetic or natural fibers,wool or any materials liable to be used for the manufacture of clothingor underclothing, active materials of any sort, wipes, patches,compresses, cottons, and cotton buds, dressings, makeup-removingsponges, masks, or any other carrier liable to come into direct contactwith the skin and scalp to enable continuous topical delivery. A morecomplete list of possible cosmetic compositions and other usuallyemployed ingredients can be found in Robinson et al. U.S. Pat. No.6,492,326 B1, which issued on Dec. 10, 2002. The text from column 5,line 35 trough column 20, line 52 and column 20, line 61 through column34, line 11 is hereby incorporated by reference. The text and claims aspublished of WO 03/028692 published on Apr. 10, 2003 and filed Oct. 1,2002 as PCT/FR02/03344 are hereby incorporated by reference.

EXAMPLES

The following nonlimiting examples are for the purpose of illustratingsome of the uses and advantages of certain embodiments of the presentinvention. Examples 1 to 4 illustrate the various possibilities forcompositions intended for cosmetic or dermopharmaceutical usescontaining oleanolic acid and/or nordihydroguaiaretic acid or thecorresponding extracts.

Examples 5 to 10 illustrate the effects of those compositions and thesynergistic effects observed with combination of the variousconstituents.

Example 1 Concentrated Mix (Premix) for the Preparation of Anti-AcneCosmetic Products

Butylene glycol 25.00 Oleanolic acid 0.03 Nordihydroguaiaretic acid 0.04PEG-60 almond glycerides 10.00 Osmocide ® (Sederma) 64.93

Example 2: Cleansing and Moisturizing Gel for Acne-Prone Skins

Carbopol ® 1342 (Goodrich) 0.3 Propylene glycol 2 Glycerol 1 White softparaffin 1.5 Cyclomethicone 6 Cetyl alcohol 0.5 Lubrajel ® MS (UnitedGuardian) 10 Triethanolamine 0.3 Oleanolic acid 0.0015Nordihydroguaiaretic acid 0.002 Osmocide ® (Sederma) 9.0 Water,preservatives, fragrance q.s. 100 g

Example 3 Care Cream for Oily Skins

Brij ® 721 (ICI) 2.4 Volpo ® S72 (Croda) 2.6 Prostearyl-15 (Croda) 8.0Beeswax 0.5 Abil ® ZP 2434 (Goldschmidt) 3.0 Propylene glycol 3.0Carbopol ® 941 (B. F. Goodrich) 0.25 Triethanolamine 0.25 Olea europaeaextract 10.0 Larrea divaricata extract 5.0 Osmocide ® (Sederma) 10.0Water, preservatives, fragrances q.s. 100 g

Example 4 Cleansing Lotion

Ethanol 1.0 Propylene glycol 2.0 Abil ® B8851 (Goldschmidt) 0.5Eumulgin ® L (Henkel) 0.6 Oleanolic acid 0.05 Water, preservatives,fragrance q.s. 100 g

Example 5 Inhibition of 5α-reductase—Enzymatic assay—In vitro

Principle and implementation of the test (adapted from Zy and Zu, 1998):

Testosterone is irreversibly metabolized by 5α-reductase to5-dehydrotestosterone (5-DHT) in the presence of the cofactor NADPH. Thereaction is monitored, at 37° C., by determining, using aspectrophotometer, the reduction in the absorbance of the reactionmedium at 340 nm, due to the conversion of NADPH to NADP.

The test thus consists in mixing testosterone, NADPH and the testproduct or its vehicle alone (control reaction). After 3 minutes toallow the system to equilibrate, the baseline absorbance of the systemis determined. Then the reaction is triggered by addition of the enzyme,5α-reductase. After 10 minutes, a second determination of theabsorbance, final absorbance, is conducted. The difference between thetwo measurements reflects the quantity of NADPH consumed. That quantityis proportional to the quantity of testosterone converted to 5-DHT.

Oleanolic acid and nordihydroguaiaretic acid were tested at variousconcentrations alone or in combination.

Results: the following table shows the percentage inhibition (mean±SEM)observed in 5 independent assays. Test product [c] % inhibitionOleanolic acid 0.01% 16.7 ± 1.1% 0.03% 31.7 ± 2.9%  0.1% 56.3 ± 2.6%NDGA 0.01%  2.1 ± 1.6% 0.04%  3.7 ± 2.9%  0.1%  3.0 ± 1.9% Oleanolicacid + 0.03% nordihydroguaiaretic acid 0.04% 68.3 ± 3.1%

These results clearly demonstrate that oleanolic acid has a stronginhibitory activity on the enzyme, 5α-reductase, since, under the aboveexperimental conditions, 56% inhibition was observed with only 0.1%.Moreover, this effect is specific since it is concentration-dependent.

Nordihydroguaiaretic acid used alone has no activity in this test.

There is strong synergy between the effects when the 2 products are usedin combination, since the inhibition observed (68%) is markedly greaterthan the sum of the individual effects observed in the same test at thesame concentrations, since the sum of the effects is only 35.4%.

Example 6 Inhibition of 5α-Reductase—HPLC Determination—In Vitro

Principle and implementation of the test: the aim of this test was toconfirm the previous result but using an HPLC analysis method. Unlike5-DHT, testosterone absorbs light at the wavelength used (245 nm). Thedecrease in testosterone peak area (which was characterized under ouroperating conditions by a retention time of 26 minutes) reflects thereduction of the concentration of the compound in the study medium andthus reflects 5α-reductase activity.

The same type of protocol as in the previous example was used, butinstead of determining the absorbances, the reaction media were analyzedby HPLC.

Results: the following table shows the percentage inhibition (mean±SEM)observed in 5 independent assays. Test product [c] % inhibitionOleanolic acid 0.01% 10.9 ± 1.4% (obtained from olive tree 0.03% 25.6 ±2.0% leaves)  0.1% 47.7 ± 3.8% Nordihydroguiararetic acid 0.01%  1.2 ±0.4% (obtained from L. divaricata) 0.04%  1.0 ± 0.9%  0.1%  0.9 ± 0.7%Oleanolic acid + 0.03% nordihydroguaiaretic acid 0.04% 61.8 ± 4.2%

The results clearly show that:

the effects observed in this example are very similar to those observedin the previous example confirming the initial hypothesis;

oleanolic acid is endowed with strong inhibitory activity vis-à-vis theenzyme, 5α-reductase, since, under our experimental conditions, 48%inhibition was observed with only 0.1% oleanolic acid.Nordihydroguaiaretic acid used alone had no activity in this test;

there is patent synergy between the effects when the 2 products are usedin combination, since the observed inhibition (61.8%) is markedlygreater than the sum of the individual effects observed in the same testat the same concentrations, since the sum of the effects is only 26.6%.

Example 7 Anti-Inflammatory Effect—In Vitro Principle and TestImplementation

It is possible to estimate a degree of inflammation by determining thequantity of mediators such as prostaglandins, leukotrienes orinterleukins.

In this example, we selected assay of interleukin 6 (IL-6) on normalhuman fibroblasts (HF) and keratinocytes (HK) in culture followingexposure to UV-B irradiation.

Broadly speaking, the cells were cultured in a conventional culturemedium (DMEMc+10% FCS) for periods of 24 hours for HF and 1 week for HK.After elimination of the buffer and 2 successive washings with phosphatebuffer solution, the cells were exposed to UV-B irradiation at astandardized energy level of 50 mJ.cm⁻².

The buffer was then rapidly eliminated and replaced by the same culturemedium as that used at the start of the experiment, but containing thetest extracts at the prerequisite concentrations or DMSO for the controlseries. After 24 hours, the culture medium was harvested and IL-6concentration determined using a standard ELISA method.

An additional series was conducted using the same protocol but withoutUV-B irradiation in order to determine the baseline level and controlthe stability of the study system. All the assays were conducted intriplicate.

The following table shows the results (mean±SEM of the percentage changevs. the tests conducted without UV radiation exposure) for 5 independentassays. HF HK Test product [c] % change % change Control — +439 ± 11%+450 ± 15% Oleanolic acid 0.01% +455 ± 14% +461 ± 16% 0.03% +437 ± 12%+444 ± 14%  0.1% +441 ± 18% +450 ± 14% NDGA 0.01% +356 ± 18% +388 ± 10%0.04% +155 ± 9%  +201 ± 10%  0.1% +87 ± 7%  +91 ± 14% Oleanolic acid +0.03% nordihydroguaiaretic 0.04% +47 ± 4% +58 ± 4% acid

The results clearly show that:

nordihydroguaiaretic acid has a remarkable anti-inflammatory effect onthe 2 cell lines studied since, under our experimental conditions (HFand HK), inhibition of 19% (+356 vs. +439%) and 14% (+388 vs. +450%) wasobserved with a nordihydroguaiaretic acid concentration of only 0.01%.Moreover, the effect was specific, since it was concentration-dependent;

oleanolic acid used alone is devoid of activity in this test;

for the 2 cell lines, there was patent synergy between the effects whenthe 2 compounds were used in combination, since 90% inhibition (+47 vs.+439%, HF) and 87% inhibition (+58 vs. +450%, HK), respectively, wereobserved. These inhibitions were markedly greater than the sum of theindividual effects observed in the same test at the same concentrations.

Example 8 Antiproliferative Effect on Keratinocytes—In Vitro

Test principle and implementation: since the quantity of DNA is constantfor each cell, the determination of the overall quantity of DNA isequivalent to measuring the number of cells used for the determination.This principle enables routine use of more refined but very cumbersomemethodologies to be avoided. Various methods have been developed on thebasis of this protocol, of which, in particular, that used herein. Whenbound to DNA with a constant and known stereochemistry, the fluorophore,Hoechst 33258, presents, first, increased fluorescence, but also anemission spectrum shift from 492 to 458 nm. By means of comparison withpre-established sets of standards, the concomitant monitoring of theabove 2 parameters enables quantification of the quantity of DNA presentin the cell samples studied.

Broadly speaking, normal human keratinocytes (HK) are cultured in aclassic culture medium (DMEMc+10% FCS) for 1 week, in the absence of thetest product (control series in order to determine the baseline cellproliferation of the system studied) or in the presence of the testproducts alone or in combination. Fluorophore Hoechst 33258 was added atthe end of the protocol before withdrawing cell aliquots for assay. Theassays were conducted in triplicate as 3 mutually independent assays.

Results: the following table shows the percentage inhibition (mean±SEM)with respect to the quantities of DNA (and hence the number of cellspresent, as observed in each of the 5 independent assays). Test product[c] % inhibition Oleanolic acid 0.01%  0.9 ± 1.0% 0.03%  1.1 ± 1.6% 0.1%  0.7 ± 0.7% NDGA 0.01% 10.2 ± 1.5% 0.04% 22.3 ± 1.9%  0.1% 29.9 ±1.7% Oleanolic acid + 0.03% nordihydroguaiaretic acid 0.04% 41.7 ± 3.4%

The results clearly show that:

nordihydroguaiaretic acid strongly inhibits keratinocyte proliferationsince, under our experimental conditions, 10% inhibition was observedwith a concentration of only 0.01% nordihydroguaiaretic acid. Moreover,the effect was specific since it was concentration-dependent;

oleanolic acid used alone was devoid of activity in this test;

there was a patent synergy between the effects when the 2 compounds wereused in combination, since the observed inhibition (41.7%) was markedlygreater than the sum of the individual effects observed in the same testat the same concentrations, since the sum was only 23.4%.

Example 9 Antibacterial Effect on Corynebacterium Acnes—In Vitro

Test principle and implementation: 1 mL of the premix in example No. 1or its constituents at concentrations equivalent to 5% of the premixwere incorporated in molten agar medium (BioMerieux) plated in a Petridish. The dish was stirred to blend the compound in the agar. 10 μL ofstudy bacteria suspension diluted to contain approximately 10⁴ and 10⁵bacteria/mL was applied to the solid agar at room temperature. Theplates were then incubated at 37° C. for 24 and 48 hours and inspectedvisually.

Five strains, Corynebacterium minutissimum, Propionibacterium acnes,Staphylococcus aureus, Staphylococcus hominis and Acinetobacteriumcalcoaceticus were studied.

Results: The following table shows the observed antimicrobial activityresults for the constituents and their combination in the cosmeticexcipient.

Oleanolic acid exerted selective antimicrobial activity against themicroorganisms P. acnes and A. calcoaceticus. Nordihydroguaiaretic acidexerted selective antimicrobial activity against A. calcoaceticus and S.hominis. OSMOCIDE® exerted selective antimicrobial activity against S.aureus, S. hominis and P. acnes, while none of the other constituentsshowed any effect.

The combination of the constituents in the premix in example 1 showedthe most complete antimicrobial activity, inactivating all the testmicroorganisms.

The strains studied are shown in the table as:

Strain A: Corynebacterium minutissimum

Strain B: Staphylococcus hominis

Strain C: Staphylococcus aureus

Strain D: P. acnes

Strain E: Acinetobacterium calcoaceticus Strains studied A B C D EBacteria/mL 10⁵ 10⁴ 10⁵ 10⁴ 10⁵ 10⁴ 10⁵ 10⁴ 10⁵ 10⁴ Butyleneglycol + + + + + + + − − − − oleanolic acid + + − − + + + − − − Butyleneglycol + + + + + + + + + + + nordihydroguaiaretic + + − − − − − − + +acid + + + + + + + + + + Butylene glycol + − − − − − − − − − − waterOSMOCIDE − − − − − − − − − − PEG-60 almond oil + water 5% premix No. 1Trichlosan (reference substance)The results at 24 h (above) were the same as those observed at 48 h.Key:‘+’ = bacterial growth,‘−’ = no bacterial growth

Example 10 Anti-Acne Effect—In Vivo

The study was conducted in 30 volunteers, with equal numbers of bothgenders, aged 14 to 20 years, selected for their acne-prone skin, i.e.presenting with at least 2 inflammatory lesions (papules, pustules,nodules) and/or 10 retentive lesions (comedo and microcyst).

The test product, the gel in example No. 2, was applied for 2 months,morning and night, after conventional cleansing of the skin. All otherproducts (topical medication, cosmetic or dermatological products) wereprohibited throughout the duration of the study.

The lesions were counted and observed by dermatologists, on the samepredetermined area of the facial skin (20 cm²), pre-treatment (T0) andafter 30 (T30) and 60 (T60) days of treatment. A significant decrease inthe lesions and a marked improvement in the state of the face wereobserved after the cosmetic treatment. The product did not induceirritation, edema or other signs of poor tolerance.

1. A method of treating acne-prone skin comprising the steps ofproviding a cosmetic or dermopharmaceutical composition comprising aneffective quantity of oleanolic acid, wherein said effective quantity isbetween about 1 ppm and about 10,000 ppm, and applying said compositionto skin in need thereof.
 2. The method of claim 1, wherein saideffective quantity of oleanolic acid is between about 10 ppm and about1,000 ppm.
 3. The method of claim 1, wherein said composition furthercomprises nordihydroguaiaretic acid.
 4. The method of claim 3, whereinsaid nordihydroguaiaretic acid is between about 1 and about 10,000 ppm.5. The method of claim 1, wherein said composition further comprises atleast one other ingredient employed in the treatment of acne,hyperseborrhea and acne-prone skin.
 6. The method of claim 5, whereinsaid at least one other ingredient employed in the treatment of acne,hyperseborrhea and acne-prone skin is selected from the group consistingof keratolytic, sebum-regulating, moisturizing and osmotic activesubstances.
 7. A method of treating skin inflammation in the skin of asubject in need thereof comprising the steps of providing a cosmetic ordermopharmaceutical composition comprising an effective quantity ofoleanolic acid, wherein said effective quantity is between about 1 ppmand about 10,000 ppm, and applying said composition to skin in needthereof.
 8. The method of claim 7, wherein said effective quantity ofoleanolic acid is between about 10 ppm and about 1,000 ppm.
 9. Themethod of claim 7, wherein said composition further comprisesnordihydroguaiaretic acid.
 10. The method of claim 9, wherein saidnordihydroguaiaretic acid is between about 1 and about 10,000 ppm. 11.The method of claim 7, wherein said composition further comprises atleast one other ingredient employed in the treatment of acne,hyperseborrhea and acne-prone skin.
 12. The method of claim 11, whereinsaid at least one other ingredient employed in the treatment of acne,hyperseborrhea and acne-prone skin is selected from the group consistingof keratolytic, sebum-regulating, moisturizing and osmotic activesubstances.
 13. A method of treating or preventing symptoms of acne orhyperseborrhea comprising the steps of providing a cosmetic ordermopharmaceutical composition comprising an effective quantity ofoleanolic acid, wherein said effective quantity is between about 1 ppmand about 10,000 ppm, and optionally nordihydroguaiaretic, and applyingsaid composition to skin in need thereof.
 14. The method of claim 13,wherein said effective quantity of oleanolic acid is between about 10ppm and about 1,000 ppm.
 15. The method of claim 13, wherein saidnordihydroguaiaretic acid is between about 1 ppm and about 10,000 ppm.16. The method of claim 13, wherein said composition further comprisesat least one other ingredient employed in the treatment of acne,hyperseborrhea and acne-prone skin.
 17. The method of claim 16, whereinsaid at least one other ingredient employed in the treatment of acne,hyperseborrhea and acne-prone skin is selected from the group consistingof keratolytic, sebum-regulating, moisturizing and osmotic activesubstances.
 18. A method of reducing 5a-reductase in the skin of asubject in need thereof comprising the steps of providing a cosmetic ordermopharmaceutical composition comprising an effective quantity ofoleanolic acid, wherein said effective quantity is between about 1 ppmand about 10,000 ppm, and administering said an amount of saidcomposition to a portion of said skin of said subject sufficient toreduce the levels of 5α-reductase in said skin of said subject.
 19. Themethod of claim 18, wherein said effective quantity of oleanolic acid isbetween about 10 ppm and about 1,000 ppm.
 20. The method of claim 18,wherein said composition further comprises nordihydroguaiaretic acid.21. The method of claim 20, wherein said nordihydroguaiaretic acid isbetween about 1 and about 10,000 ppm.
 22. The method of claim 18,wherein said composition further comprises at least one other ingredientemployed in the treatment of acne, hyperseborrhea and acne-prone skin.23. The method of claim 22, wherein said at least one other ingredientemployed in the treatment of acne, hyperseborrhea and acne-prone skin isselected from the group consisting of keratolytic, sebum-regulating,moisturizing and osmotic active substances.